To generate measures of the sensitivities of three independent batches and one derivative of the MCF 10A cell line to 8 small molecule perturbagens, we treated cells with single drugs over a minimum 9-point dilution series using SQRT(10) dilutions centered around the GR50 but not exceeding 10 µM and then measured cell number after three days of drug exposure.

Assay Protocol:

1. Cells in mid-log phase of the growth cycle for three independent batches of MCF 10A and for one batch of an H2B-mCherry-expressing derivative of MCF 10A were each plated at 750 cells/well in 60 µL of complete growth medium (DMEM: F-12 + 5% (v/v) horse serum + 10 µg/mL human insulin + 20 ng/mL rhEGF + 100 ng/mL Cholera toxin + 0.5 µg/mL Hydrocortisone) in triplicate 384-well plates.
2. The plated cells were grown for 24 hours at 37°C in the presence of 5% CO2.
3. Two of the plates were treated with the indicated small molecules by direct dispensing of DMSO stock solutions to the indicated concentrations into 60 µL of media using an HP D300 Digital Dispenser and then were allowed to grow for 3 additional days.
4. Immediately after treatment of the two plates, the untreated third plate was fixed and stained by adding 20 µL of staining solution (1:1000 LIVE/DEAD Far Red Dead Cell Stain (Thermo Fisher Scientific, catalog #L-34974), 2 µM Hoechst 33342 (Thermo Fisher Scientific, catalog #62249), 10% OptiPrep (Sigma-Aldrich, catalog #D1556-250ML) in PBS). The plate was incubated for 30 min at room temperature, 90 µL of supernatant per well was removed and replaced with the same volume of PBS, and the plate was imaged as described in step 6.
5. On day 3 the treated plates were fixed, stained, and prepped for imaging as described in step 4.
6. The plates were scanned with a PE Operetta high-throughput plate scanner.
7. Nuclei for cells that were alive at the time of fixation were quantified using Columbus software.
8. Nuclei counts were normalized to DMSO-treated controls on the same plate to yield relative cell count and normalized growth rate inhibition (GR) values for each technical replicate for each cell line batch / small molecule / small molecule concentration combination. Relative cell count is the measured cell count for a given treatment divided by the 50%-trimmed mean of the cell count of the DMSO-treated control wells on the same plate. Consistent with the methods reported in Hafner et al. (2016) (PMID: 27135972), normalized growth rate inhibition (GR) values were calculated according to the following formula: 2^[log2(x(c)/x0)/log2(xctrl/x0)]-1 where x(c) is the measured cell count after a given treatment, x0 is the 50%-trimmed mean of the cell count from the day 0 untreated plate grown in parallel until the time of treatment, and xctrl is the 50%-trimmed mean of the cell count of the DMSO-treated control wells on the same treated plate. Data for three complete biological replicates were analyzed (HMS LINCS Datasets #20278-20280).
9. Nuclei counts for all technical replicates within a single biological replicate were averaged to yield mean relative cell counts and the mean normalized growth rate inhibition (GR) value for each cell line batch / small molecule / small molecule concentration combination within each biological replicate (HMS LINCS Datasets #20281-20283).
10. Again within each biological replicate, mean normalized growth rate inhibition (GR) values for a given cell line batch / small molecule combination across all tested concentrations were fitted to a sigmoidal curve to extract the GR50, GRmax, Hill coefficient, r2, and GR_AOC values (HMS LINCS Datasets #20284-20286).